P.I. S. Scarlata Phospholipase C-b (PLC?) enzymes are activated by G proteins in response to agents such as hormones and neurotransmitters. PLC? activity causes an increase in intracellular calcium which ultimately leads to profound changes in the cell. Our lab has focused on the mechanism through which G proteins activate PLC? on the molecular level. However, in cultured cells, we find additional and unexpected mechanisms of regulation. First, membrane domains called caveolae can control the duration of PLC? activation by selectively sequestering G protein activators. 1 - In AIM 1, we will use live cell imaging and state of the art fluorescence methods to determine the mechanisms through which caveolae prolongs PLC? - mediated calcium signals and directs signals along specific cellular pathways. 2- We have found that PLCb localizes to the nucleus and cytosol as well as the plasma membrane where its G protein activators are found. We have identified a novel protein partner of PLC? translin-associated protein X (TRAX). TRAX and its partner translin are major components of the siRNA machinery regulating the cellular levels of proteins. In AIM 2, we will determine the ability of TRAX to deliver PLC? from the nucleus to the plasma membrane upon G protein activation. In parallel, we will determine whether PLC affects translin-TRAX function. 3- The ability of PLC? to generate signals depends on its cellular concentration. We have found that the synucleins can binds to and stabilize PLC? in cells. Alpha-synuclein (AS) is a small unfolded protein of unknown function and is a major component of neurodegenerative plaques. The presence of AS greatly increases PLC? levels, and we find that down-regulation of PLC??causes AS aggregation. Also, gamma- synuclein (GS), the protein encoded by the breast cancer susceptibility gene 1, appears to increase PLCb -mediated migration and cell invasiveness thereby promoting breast cancer. In AIM 3 we will better define PLC? - synuclein interactions and develop reagents that prevent AS aggregation and reduce GS-induced breast cancer phenotype. PUBLIC HEALTH RELEVANCE: P.I. Scarlata, S. Project Narrative In cells, enzymes are subject to other forms of regulation that are distinct and not seen in vitro. Here, we will continue our studies of the activation of phopsholipase C-2 (PLC2) focusing on its cellular regulation which may be applicable to other enzymes. Importantly, the link between PLC2 and synucleins in promoting neurodegenerative disease and breast cancer will be explored to develop more effective therapeutics.